AEM Accepts, published online ahead of print on 26 July 2013 Appl. Environ. Microbiol. doi:10.1128/aem.01745-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 3 Antimicrobial resistance in indicator Escherichia coli from free-ranging livestock and sympatric wild ungulates in a natural environment (NE Spain) 4 5 6 7 8 9 10 11 12 13 14 15 16 Navarro-Gonzalez N 1*, Porrero MC 2, Mentaberre G 1, Serrano E 1,3, Mateos A 2, Domínguez L 2, Lavín S 1. 1 Servei d Ecopatologia de Fauna Salvatge (SEFaS), Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona (UAB), Bellaterra, Barcelona, Spain. 2 Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense (UCM), Madrid, Spain. 3 Estadística i Investigació Operativa, Departament de Matemàtica, Universitat de Lleida (UdL), Lleida, Spain. * corresponding author. E-mail adress: norit85@gmail.com Running title: AMR in extensive farming and sympatric wildlife 17 18 19 1
20 Abstract: 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Antimicrobial resistance was assessed in indicator E. coli in free-ranging livestock and sympatric wild boar (Sus scrofa) and Iberian ibex (Capra pyrenaica) in a National Game Reserve in NE Spain. The frequency of antimicrobial resistance was low (0 to 7.9%). However, resistance to a third-generation cephalosporin and fluoroquinolones was detected. 36 37 38 2
39 Short communication 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Antimicrobial resistance (AMR) may compromise the treatment of severe human diseases (1), and thus monitoring and reporting its occurrence is a priority for health surveillance agencies worldwide. This phenomenon has been partly associated with the use of antimicrobial agents in intensive animal food production (2); in fact, a lower occurrence of resistant bacteria has been repeatedly observed in extensive or organic farming systems when compared to intensive ones (3-6). Moreover, many studies have found similarities in the patterns of resistance in bacterial isolates from livestock and small fauna, e.g. rodents (7, 8), insects (7, 9) or birds (9) from the farm settings. Thus, we were interested in determining whether wild ungulates in close contact with free-ranging livestock carry indicator bacteria with similar resistance profiles. Indicator (commensal) Escherichia coli is suitable for such a study, since it is common in animal feces and provides information on resistance in a population (1). For this purpose, we sampled both wild ungulates (wild boar - Sus scrofa - and Iberian ibex - Capra pyrenaica) and free-ranging livestock sharing habitat in a game reserve in NE Spain. The use of antimicrobials in this study area can be ruled out and human activities, and thus, selective pressure, are reduced. Therefore, we expect E. coli from these host populations to be almost free of antimicrobial resistance. 58 59 The study area is located within the National Game Reserve and Natural Park Ports de Tortosa i Beseit (NGR hereafter), in northeastern Spain. Wildlife and livestock share 3
60 61 pastures in some canyons in the study area. See (10) for further information on the area and the livestock presence. 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 One hundred and forty-three (n= 143) individual fecal samples were obtained from hunter-harvested wild boars during the regular hunting season (October to January) from 2009 to 2011. Forty-six individual fecal samples (n = 46) were obtained from cattle (5 herds, 380 head in total) and four from the only horse herd in the NGR (32 head). Feces were collected and stored in a sterile container and refrigerated until being sent to the laboratory within the subsequent 24 hours. One hundred and eighty-four (n =184) Iberian ibexes were either hunter-harvested (n =154) or captured (n =30) from 2009 to 2011. Due to the characteristics of the hunting method, fecal samples had to be stored at -18 ºC until being sent to the laboratory. E. coli are known for their cold-shock response (11) and thus, we can assume that the isolation of this microorganism from feces is not highly affected by storage at this temperature. In total 25g of faeces were diluted in buffered peptone water (225ml). Once diluted, one loop was cultured on MacConkey agar (direct plating) at 37ºC for 18-20h. One compatible colony per plate was selected and confirmed by PCR (12). This confirmed colony of indicator E. coli (i.e. one clon per animal) was tested for antimicrobial susceptibility (13, 14). Table 1 shows the antimicrobial agents and epidemiological cutoff values used to report microbiological resistance (1). 80 81 82 All isolates from livestock were tested for antimicrobial resistance (n = 42, from which: 38 from cattle, 4 from horse). A selection of E. coli isolates from wildlife was performed to spatially represent the whole study area; therefore, one isolate was 4
83 84 selected per location and hunting session for wild boar and Iberian ibex (altogether, 63 and 89 isolates, respectively). 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 For the comparison of our data with that from intensively-reared livestock, Table 2 shows the frequencies of resistant E. coli in cattle and pigs from the European Union (1) and Spain (VAV Network data, personal communication). Frequencies of resistance in E. coli were compared between host species with the Fisher s exact test and the significance level set at α = 0.05. The p-values obtained from multiple comparisons were adjusted with the strict Bonferroni correction. The statistical analyses were performed with R Software (15). Eight wild boars (12.7%), 4 cows (10.53%) and 3 Iberian ibexes (3.37%) and no horse were carriers of E. coli resistant to the antimicrobial agents tested (7.65% of the total tested samples). These frequencies were not statistically different (wild boar- cattle: adjusted p-value= 1, wild boar- Iberian ibex: adjusted p-value= 0.15, cattle- Iberian ibex: adjusted p-value=0.63). No isolate resistant to colistin, amoxicillin-clavulanate, cefoxitin, amikacin, apramicin, imipenem, aztreonam, gentamicin, ceftazidime, chloramphenicol or florfenicol was found. Table 2 shows the percentage of isolates from each host group showing resistance to the rest of antimicrobial agents tested. Frequencies of resistance ranged from 0 to 7.9%. Cattle from the NGR had significantly lower (p < 0.05) frequency of E. coli resistant to sulfamethoxazole, ampicillin, tetracycline, streptomycin and trimethoprim than intensively-reared cattle from Spain (Table 2). The same resistance profile was rarely detected more than once (Table 3). 104 105 Frequencies of AMR were less than 10%, which is defined by the EFSA (1) as low resistance level. In the literature great variations are observed in AMR depending on 5
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 the species, the ecosystem and the geographic location. In some cases, this variation has been connected to the presence of farms (16) or interactions with farm waste (17), livestock rates (18), human proximity (19) or human density (20). Skurnik et al. (20) reported a higher resistance score in extensively-reared farm animals compared to wildlife from the same area. However, free-ranging livestock appears not to be a main source of AMR in our study area since the resistance frequency was not higher in E. coli from livestock. In general, these livestock show resistance levels lower than those reported by the Spanish VAV Network for intensively-reared livestock. Other potential sources of AMR may exist in the study area. Indeed, multidrug-resistant bacteria have been isolated from a range of wild animals with no known previous exposure to antimicrobial agents, a fact that suggests that resistance is not confined to the ecological niche where it emerged (21). Resistance to a third generation cephalosporin (cefotaxime) and to fluoroquinolones was found. These agents are listed as critically important antimicrobials for human medicine by the WHO (22) and the carriage of resistant bacteria by wildlife is of concern for public health. In fact, wild boars have been found to be carriers of cefotaxime-resistant E. coli in countries as diverse as Poland (23), the Czech Republic (24) and Portugal (25). In spite of a general low frequency of AMR, this shows that protected natural environments are not exempt from the introduction of anthropogenic AMR. Furthermore, this study shows that livestock in an extensive farming system are not important contributors to the AMR in E. coli in co-habiting wild ungulates. 128 Acknowledgments 6
129 130 131 132 133 134 135 136 137 138 139 140 141 142 We express our gratitude to the Departament d Agricultura, Ramaderia, Pesca, Alimentació i Medi Natural of the Generalitat de Catalunya for supporting our research activity. We are also very thankful to the staff of the National Game Reserve and the Natural Park Els Ports de Tortosa i Beseit for its valuable help in the sampling and gathering information on the location of the herds. This work was partially supported by the Ministry of Science and Innovation within the Program of Interaction between wild animals and livestock (FAU2008-00021) and by the Autonomous Community of Madrid, Spain (S0505 AGR-0265; S2009 AGR-1489). Authors also wish to thank the technicians M. Carmen Comerón, Nisrin Maasoumi and Lorena del Moral for their excellent work. N. Navarro-Gonzalez was supported by the FPU program from the Ministerio de Educación (Spain) and E. Serrano by the Beatriu de Pinós programme (BP-DGR 2011) of the Catalan Science and Technology System (Spain). 7
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235 Table 1. Antimicrobial agents and epidemiological cut-off values. 236 Antimicrobial agent Epidemiological cut-off value Reference 237 Disk diffusion Amoxicillin-clavulanate 17 mm EUCAST Cefoxitin 19 mm EUCAST 238 239 Amikacin 18 mm EUCAST 240 Apramicin 20 mm Rosco diagnostica Imipenem 24 mm EUCAST Aztreonam 27 mm EUCAST Broth microdilution Sulfamethoxazole 64 µg/ml EFSA Gentamicin 2 µg/ml EFSA Ampicillin 8 µg /ml EFSA Ciprofloxacin 0.064 µg /ml EFSA Cefotaxime 0.25 µg /ml EFSA Ceftazidime 0.5 µg /ml EFSA Tetracycline 8 µg /ml EFSA Streptomycin 16 µg /ml EFSA Trimethoprim 2 µg /ml EFSA Chloramphenicol 16 µg /ml EFSA Florfenicol 16 µg /ml EFSA Kanamycin 8 µg /ml EUCAST Nalidixic acid 16 µg /ml EFSA Colistin 2 µg /ml EFSA 241 242 243 244 245 246 247 248 249 250 251 252 EFSA: EFSA Journal 2012; 10:2742. EUCAST: www.srga.org/eucastwt/wt_eucast.htm 253 13
254 Table 2. Frequencies of resistant E. coli in each host species of the National Game Reserve, and from cattle from intensive rearing in Spain. Antimicrobial agents to which pansusceptibility in the NGR was found are referred in the text. National Game Reserve Spain Antimicrobial agent Wild boar Iberian ibex Cattle Cattle a 255 Ciprofloxacin 3.2 0 7.1 3.1 Sulfamethoxazole 6.3 1.1 2.3 35.2 Ampicillin 4.8 1.1 2.3 15.6 Cefotaxime 1.6 0 0 0 Tetracycline 7.9 3.3 2.3 48.8 Streptomycin 4.8 1.1 2.3 35.5 Trimethoprim 3.2 1.1 0 17.6 Kanamycin 6.3 0 0 2.7 Nalidixic acid 1.6 0 4.7 3.1 In bold, statistically significant differences between free-ranging cattle from our study area and intensively-reared cattle from Spain (p < 0.05) a VAV Network data, personal communication. Downloaded from http://aem.asm.org/ on May 3, 2018 by guest 14
256 Table 3. Phenotypic profile of the resistant E. coli strains isolated from the three host species. Host species (number of isolates) Resistance profile Wild boar CIPR,SMX,AMP,CEFOT,TET,NAL Wild boar SMX,AMP,TET,STR,KAN Iberian ibex SMX,AMP,TET,STR,TMP Wild boar SMX,TET,STR,TMP,KAN Wild boar CIPR,AMP,STR Wild boar SMX,TET,TMP Cattle SMX, TET, STR Cattle CIPR, AMP, NAL Cattle CIPR,NAL Cattle CIPR Wild boar (2) KAN Iberian ibex (2), Wild boar TET CIPR: Ciprofloxacin, SMX: Sulfamethoxazole, AMP: Ampicillin, CEFOT: Cefotaxime, TET: Tetracycline, NAL: Nalidixic acid, STR: Streptomycin, KAN: Kanamycin, TMP: Trimethoprim. Downloaded from http://aem.asm.org/ on May 3, 2018 by guest 15